P53-Induced Protein with a Death Domain (PIDD): Master of Puppets&Quest

REVIEW P53-induced protein with a death domain (PIDD): master of puppets?

FJ Bock, L Peintner, M Tanzer, C Manzl and A Villunger

P53-induced protein with a death domain (PIDD) has been described as primary p53 target gene, induced upon DNA damage. More than 10 years after its discovery, its physiological role in the DNA damage response remains enigmatic, as it seems to be able to execute life--death decisions in vitro, yet genetic ablation in mice failed to reveal an obvious phenotype. Nonetheless, evidence is accumulating that it contributes to the ﬁne-tuning of the DNA-damage response by orchestrating critical processes such as caspase activation or nuclear factor kB translocation and can also exert additional nuclear functions, for example, the modulation of translesion synthesis. In this review, we aim to integrate these observations and propose possible unexplored functions of PIDD.

Oncogene (2012) 31, 4733--4739; doi:10.1038/onc.2011.639; published online 23 January 2012 Keywords: PIDD; Caspase-2; RAIDD; p53; NFkB

INTRODUCTION PIDD DISCOVERY Genotoxic stress poses a dangerous threat to the integrity of PIDD was first described in the year 2000 by two research groups the genome. It is therefore not surprising that cells have independently. Telliez et al.6 identified a protein dubbed LRDD evolved multiple mechanisms to adequately process sustained (leucine-rich repeats and death domain containing protein) in a DNA damage.1 One central mediator of the cellular signalling bioinformatics search for proteins with a DD, similar to the DD of response after DNA damage is the tumor suppressor p53.2,3 receptor interacting protein (RIP-1). They already noted that LRDD/ In unstressed conditions, p53 is rapidly ubiquitinated by Mdm2 PIDD is processed within the cell (see below) and detected and subsequently degraded via the proteasomal pathway. interactions with other (see Table 1), but not all, DD containing However, in response to DNA damage p53 is posttranslationally proteins. The second group5 used differential display of an modified at various residues, for example, by phosphorylation erythroleukemia cell line transfected with a temperature sensitive or acetylation that prevents or fine-tunes its ubiquitination p53 mutant (p53ts A135V). One gene upregulated after culturing and subsequent degradation.4 These events lead to stabilization the cell line at the permissive temperature was named PIDD, a and translocation of p53 into the nucleus, where it induces splice variant of which being equivalent to LRDD. The promoter the transcription of numerous target genes containing p53- for the PIDD gene contained a bona fide p53-binding site, and the binding sites in their promoter. Those target genes can have such messenger RNA (mRNA) itself is strongly induced after over- different outcomes as apoptosis (for example, Noxa, Bax expression of functional p53 or g-irradiation (IR). No induction was and Puma), cell cycle arrest and repair (for example, p21 and observed in several cell types deficient for p53, which additionally GADD45), induction of autophagy (for example, DRAM and had lower basal levels of PIDD mRNA. Overexpression of PIDD Sestrin2) or inhibition of glucose metabolism (for example, TIGAR). reduced cell proliferation and increased apoptosis comparable to The exact mechanisms that regulate the differential expression of p53 overexpression. Furthermore, antisense oligonucleotides specific target genes are not definitely established until now. targeting PIDD mRNA for degradation led to reduced apoptosis Transcription and subsequent translation of p53 target genes in response to g-IR.5 leads to arrest of the cell cycle and repair of the DNA damage and, if the damage is too severe and can not be repaired properly, cell death mediated mainly via the intrinsic Bcl-2-regulated apoptotic PIDD ISOFORMS pathway. The human PIDD gene is located on chromosome 11 and contains One p53 target gene that can modulate the decision between 16 exons. The full length PIDD protein (isoform 1) is made up by cell cycle arrest and apoptosis in response to genotoxic stress 910 amino acids and contains seven N-terminal leucine-rich repeat downstream of p53 is the p53-induced protein with a death domains, followed by two ZU5 domains and a C-terminal DD. 5,6 domain (PIDD). PIDD was first described in the year 2000 and Several other isoforms have been described:11--13 isoform 2 contains a leucine-rich repeat domain at the N-terminus followed contains a deletion between amino acids 705 and 721; isoform by two ZU5 domains and a death domain (DD) at the C-terminus. 3, also known as LRDD, lacks the first 147 amino acids and harbors Over the last decade, there have been numerous publications a deletion between amino acids 580 and 590; isoform 4 lacks reporting on possible functions for PIDD, none of them entirely the first 314 amino acids, and therefore the leucine-rich convincing, summarized and discussed in this review. repeat domain; isoform 5, which has so far only been identified

Division of Developmental Immunology, Biocenter, Innsbruck Medical University, Innsbruck, Austria. Correspondence: Professor Dr A Villunger, Division of Developmental Immunology, Biocenter, Innsbruck Medical University, Fritz-Pregl-Strasse 3, A-6020 Innsbruck, Austria. E-mail: [email protected] Received 10 November 2011; revised 6 December 2011; accepted 12 December 2011; published online 23 January 2012 PIDDosomes and the DNA damage response FJ Bock et al 4734 Table 1. PIDD interaction partners

PIDD fragment Interaction partner Identiﬁed by Effect References

PIDD-FL HSP90 Co-IP and MS Interaction enables PIDD cleavage and activation. Tinel et al.7 PIDD-FL p23 Co-IP and MS PIDD-C HSP70 Co-IP and MS Unknown Tinel et al.7 PIDD-C RIP-1 Co-IP The RIP-1-PIDDosome leads to the sumoylation Janssens et al.8 of NEMO and to subsequent activation of NFkB. PIDD-C Nemo Co-IP PIDD-C PCNA Co-IP and MS PIDD displaces p21 from PCNA to enable translesion Logette et al.9 synthesis repair in response to UV irradiation. PIDD-CC RAIDD Co-IP The RAIDD-PIDDosome can activate caspase-2. Tinel and Tschopp10 PIDD-CC Caspase-2 Co-IP PIDD-C and Nucleolin GST pulldown, Co-IP Unknown Pick et al.11 PIDD-CC and Y-2H PIDDa MADD Co-IP Unknown Telliez et al.6 PIDDa FADD Co-IP Unknown Telliez et al.6 Abbreviations: FADD, Fas-Associated protein with Death Domain; GST, glutathione-S-transferase; IP, immunoprecipitation; MADD, MAP-kinase activating death domain; MS, mass spectrometry; PCNA, proliferating cell nuclear antigen; PIDD, P53-induced protein with a death domain; RIP-1, receptor interacting protein 1. aUnclear at present which fragment constitutes the minimal unit for binding, that is full length, CC- , C- or N-terminal fragment.

with bioinformatic methods12 and was not further investi- PIDD-CC fragment. However, in thymocytes or primary keratino- gated biologically, contains only the two ZU5 domains and is cytes, the CC fragment appears to be the dominant form alternatively spliced giving rise to a frameshift and therefore a detectable, implicating that processing of PIDD can be the default different C-terminus (Figure 1a). program, even in the absence of genotoxic stress.9,16 Another Whereas isoforms 1 and 3 are expressed in all tissues examined, report shows additional processing products upon PIDD or LRDD with the exception of skeletal muscle, isoform 2 mRNA is only overexpression; however, the exact nature of those fragments detectable in liver, pancreas and leukocytes. Isoform 4 is has not been investigated in more detail,11 but N-terminal expressed in all tissues including skeletal muscle, but not in the protein sequencing may help to identify the nature of these heart.12,13 The lack of reliable antibodies recognizing and PIDD-derived polypeptides. discriminating endogenously expressed mouse PIDD isoforms, So how is cleavage regulated? PIDD can be part of a complex however, make validation of these ﬁndings at the protein level containing various molecular chaperones like HSP70, HSP90 or difﬁcult. p23, where HSP90 and p23 only interact with full length PIDD in Despite initially being described as a gene upregulated in the cytoplasm.17 Accordingly, HSP90 is required for PIDD response to DNA damage in a p53-dependent manner, not all processing into the PIDD-N and PIDD-C fragments, presumably genotoxic stressors induce PIDD. Surprisingly, PIDD mRNA levels of by facilitating a conformation of PIDD that is favorable for the isoforms 1 and 2 remain unchanged in response to ultraviolet (UV) cleavage reaction. After processing, HSP90 is released from PIDD irradiation or treatment with etoposide or doxorubicin, whereas and the latter can participate in complex formation with various isoform 3 was induced after UV and etoposide but downregulated interaction partners (see below). HSP90 seems to be required for after doxorubicin.12 Of note, basal levels of PIDD protein, both processing to PIDD-C as well as PIDD-CC, although it is at presumably isoform 1 or its autoprocessing products (see below), present unclear how exactly processing to PIDD-CC is regulated by can also be detected in cell lines lacking functional p53, such as in HSP90, as both PIDD-CC and HSP90 have been shown not to p53À/À HCT116 colon cancer cells, indicating that basal protein interact with each other. One possibility would be the involve- expression does not rely on this transcription factor.12 ment of HSP70, which is able to bind both PIDD-C and -CC, for the second processing step, however this has not yet been tested experimentally. PIDD PROCESSING A further mode of regulation of PIDD processing occurs PIDD is involved in both pro- and anti-apoptotic pathways (see posttranscriptionally via generation of the aforementioned below), accordingly there should be a means to regulate isoforms by differential splicing. Isoform 2 carries a small deletion, participation of PIDD in one or the other. Surprisingly, the which includes the second HFS motif, and is therefore only able to decision which path to take is imprinted (at least in part) within be processed to PIDD-N and PIDD-C, but not to the PIDD-CC 12 PIDD itself. PIDD contains two conserved ‘HFS motifs’, also present fragment. in the nuclear pore protein Nup98 and ZUD/Unc5cl, a positive PIDD is mainly localized in the cytoplasm, but can also be 8 regulator of nuclear factor (NF)kB signalling, related to PIDD.14 present in the nucleus. In response to DNA-damaging agents or Similar to Nup98,15 PIDD uses an intramolecular reaction to cleave heat shock, PIDD can translocate to the nucleus where it localizes 11 the peptide bond between the phenylalanine F445 and the serine to nucleoli and is able to interact with the nucleolar protein S446, leading to the generation of the fragments PIDD-N and nucleolin or chromatin, either directly or via interaction with DNA 9 PIDD-C.7 Cleavage at a second HFS site at serine S588 within PIDD- repair proteins, which remains to be determined. C leads to the generation of the PIDD-CC fragment (see Figure 1b). PIDD-N contains the leucine-rich repeat domain and one ZU-5 domain, the biological function of this polypeptide remains THE ROLE OF PIDD IN CASPASE-2 ACTIVATION AND unknown. The PIDD-C fragment is composed of one ZU-5 domain APOPTOSIS and the DD, whereas the PIDD-CC fragment only harbors the DD. Various binding partners of PIDD have been described As the full length PIDD protein is hardly detectable in most (see Table 1), implicating PIDD in different cellular signalling cell types, it is believed that it is rapidly processed to PIDD-C. pathways (see Figure 2). The most studied interaction of PIDD The second cleavage occurs in response to high or prolonged is with caspase-2 and RIP-associated ICH1/CED3-homologous genotoxic stress, therefore leading to accumulation of the protein with death domain (RAIDD) in a complex termed the

Oncogene (2012) 4733 --4739 & 2012 Macmillan Publishers Limited PIDDosomes and the DNA damage response FJ Bock et al 4735

Figure 1. Generation of PIDD isoforms by splicing or proteolysis. (a) Currently known PIDD isoforms. Isoform 1 contains seven leucine-rich repeat (LRR) domains at the C-terminus followed by two ZU5 domains and an N-terminal DD. In isoform 2, the ﬁrst 146 amino acids as well as amino acids 580--590 are deleted. Isoform 3 harbors a deletion of amino acids 705--721. Isoform 4 is composed of the two ZU5 and the DD, whereas isoform 5 has not yet been experimentally validated. (b) PIDD processing products. Full length PIDD-FL is processed at S446 to give rise to PIDD-N and PIDD-C. PIDD-C can be further autoproteolytically cleaved at S588, generating PIDD-CC.

Figure 2. Cellular responses reported to engage PIDD. PIDD is induced in response to DNA damage in a p53-dependent manner, and depending on the type and severity of the damage is recruited to different complexes. A low amount of DNA damage leads to autoproteolytical cleavage of full length PIDD to the PIDD-C form. This fragment associates with NEMO and receptor interacting protein (RIP-1), thereby facilitating postranslational modiﬁcations on NEMO leading to activation of NFkB. After more severe genotoxic stress, PIDD-C is further autoproteolytically cleaved to the PIDD-CC fragment, which can bind to RAIDD and caspase-2, which is subsequently processed and activated. In response to UV-IR, PIDD-C can displace p21 from proliferating cell nuclear antigen (PCNA), thereby facilitating recruitment of the translesion synthesis repair machinery.

& 2012 Macmillan Publishers Limited Oncogene (2012) 4733 --4739 PIDDosomes and the DNA damage response FJ Bock et al 4736 PIDDosome.10 Caspases are cysteine-driven aspartases involved in CD95 expression and to induce the formation of a complex several cellular pathways, including cell death, inflammation, involving caspase-2, caspase-8, FADD and CD95 that may migration or differentiation.18 Apoptotic caspases in mammals can orchestrate its activation.26 be grouped into initiator caspases (8, 9 and 10) and effector An alternative cell death pathway engaging caspase-2 has caspases (3, 6 and 7). Initiator caspases become activated upon been described based on experiments with zebrafish embryos.27 hetero- or homodimerization in high molecular weight complexes In this model, caspase-2 is selectively activated in response to formed in response to apoptotic stimuli like the death-inducing DNA damage in p53-mutated zebrafish embryos if checkpoint signalling complex (DISC) for caspases-8 and -10 or the apopto- kinase 1 is inhibited simultaneously. This treatment apparently some for capsase-9. Once activated and processed within these leads to apoptosis of p53-deficient cells, frequently resisting DNA complexes, the initiator caspases cleave and thereby activate the damage-induced killing, in a caspase-2-dependent manner. It will effector caspases. Of note here, processing/cleavage of initiator be interesting to investigate whether PIDD and/or RAIDD are caspases can also occur as a secondary event by effector caspase- involved in this specific pathway of caspase-2 activation and, mediated proteolysis, and is therefore not equivalent to activation. if so, how they communicate with the DNA-damage response Hence, detection of initiator caspase fragments by western machinery. blotting is not indicative for their activation that requires Surprisingly, despite the compelling evidence for a participation proximity-induced dimerization.19,20 of the PIDDosome in cell death induced by various apoptotic Caspase-2 possesses some unique features.21 As it contains a substances, both caspase-2, RAIDD as well as PIDD knockout caspase recruitment domain (CARD) in its long prodomain, it can mouse models do not support a critical role of the PIDDosome in structurally be classified as initiator caspase. However, in contrast developmental or stress-induced apoptosis.16,28,29 Furthermore, to the other initiator caspases, caspase-2 is not able to cleave activation of caspase-2 is proficient in cells lacking PIDD, indicating effector caspases under physiological conditions. In addition, that other pathways for caspase-2 activation are likely to exist. In caspase-2 is the only caspase that can be detected within the addition, both PIDD- and RAIDD-deficient cells were able to induce nucleus, either constitutively, or upon induction of DNA damage, the formation of high molecular weight complexes containing although its precise function there is still under discussion. caspase-2.16 In contrast, the caspase-2 processing observed As mentioned above, initiator caspases need high molecular was dependent on mitochondrial outer membrane permeabiliza- weight complexes for activation, and for caspase-2 this complex tion and effector caspase activation, placing caspase-2 down- was found to involve the proteins RAIDD and PIDD.10 Within this stream in the apoptotic cascade. Conflicting data regarding a complex, RAIDD acts as an adaptor between PIDD and caspase-2, rate-limiting role for the PIDDosome in cell death triggered by making use of its CARD domain to bind the CARD domain of agents disrupting microtubule dynamics, such as vincristine or caspase-2 and binding the DD of PIDD with its own DD. This paclitaxel, has been reported. One study reported cell death in complex is formed spontaneously upon incubation of cell extracts primary low passage mouse embryonic fibroblasts (MEF) that was at 37 1C and leads to processing and activation of caspase-2. dependent on all three PIDDosome components,30 yet our own Furthermore, RAIDD seems to be the only CARD domain-contain- observations in primary as well as SV40-immortalized MEF lacking ing protein capable of binding caspase-2, and RAIDD only caspase-2 or PIDD do not support these findings.16 A similar interacts with caspase-2 among all CARD domain-containing discrepancy appears to exist in MEF transformed with different caspases. However, RAIDD may not interact exclusively with oncogenes and exposed to g-IR.16,31 Although the mode of caspase-2 and PIDD, as it was initially found as an interaction immortalization (E1a/Ras vs SV40 large T antigen) may well have partner of RIP-1.22 an impact on the DNA damage response, the different findings Several reports describe PIDDosome formation in response to reported for primary MEF exposed to microtubule poisons remain different death-inducing conditions. Transient global cerebral currently unexplained. ischemia in hippocampal CA1 pyramidal neurons in rats leads to One phenotype that has been reported for caspase-2-deficient increased levels of the PIDD-CC fragment, PIDDosome formation mice was an increased sensitivity of oocytes after genotoxic and processing of caspase-2.23 Cell death accompanied with stress.29 However, this sensitivity was not observed in PIDD- ischemia/reperfusion injury in the brain seems to be dependent deficient oocytes, again indicating a PIDD-independent function on PIDD, as in vivo knockdown of PIDD decreases apoptosis as well for caspase-2,28 whereas the role of RAIDD in oocytes cell death as caspase-2 processing. Of note, recent data suggest that PIDD, in has not been investigated so far. contrast to caspase-2 and RAIDD, is not required for cell death Overall, these studies suggest that caspase-2 can be a induced by b-amyloid, a neurotoxic protein involved in Alzheimers secondary target of other executioner caspases where it might disease (C. Troy et al., submitted). act as part of an amplification loop rather than an essential Treatment with the histone deacetylase inhibitor LBH589 was initiator caspase. However, in certain tumor cell types, frequently recently shown to trigger caspase-2 processing in adult T cell lacking p53, caspase-2 can be activated upstream of mitochondria, leukemia/lymphoma cell lines and patient-derived cell samples.24 but this activation does not strictly depend on PIDD. Consistently, In this study, knockdown of PIDD did not cause differences in the pro-apoptotic function of PIDD may engage other effectors tumor cell survival upon LBH589 treatment. However, both RAIDD than caspase-2 for cell killing.32 and caspase-2 seem to be essential for cell death induction and able to act upstream of caspase-9. Additionally, whereas caspase-2 cleavage was still observed after knockdown of PIDD, it was PIDD IN NFjB ACTIVATION AND CELL SURVIVAL impaired after knockdown of RAIDD, yet formal evidence that DNA damage not necessarily leads to programmed cell death. caspase-2 is indeed the most apical caspase activated in this Several mechanisms have evolved to allow cells to repair the setting was not provided. sustained damage in order to avoid excessive cell death or the Another chemotherapeutic drug shown to induce caspase-2 elimination of cells that are not irreparably damaged or processing is fluorouracil, a pyrimidine analog. Fluorouracil- paramount to maintain organ function, such as stem cells. Next induced apoptosis is dependent on p53 and caspase-2, which to the DNA repair machinery, an important prosurvival pathway is in this setting reportedly activated before caspase-3.25 induced by genotoxic stress is the one leading to activation of However, the observed caspase-2 processing again seems to be NFkB.33,34 Although NFkB appears to exert its most prominent role independent of the PIDDosome, as knockdown of PIDD or in the immune system,35 where it participates in the host defence RAIDD had no influence on apoptosis. Although the exact by inducing the expression of various mediators of inflammation, mechanism is not clear, fluorouracil was shown to upregulate NFkB has also been demonstrated to be activated in response to

Oncogene (2012) 4733 --4739 & 2012 Macmillan Publishers Limited PIDDosomes and the DNA damage response FJ Bock et al 4737 DNA damage, and PIDD seems to be an essential component of lacking, because all known components of DNA damage-induced this event. The aforementioned PIDD-C fragment can form a NFkB activation have additional roles, either in the DNA repair complex with NEMO (NFkB essential modulator), a component of response or in DNA damage-independent NFkB activation making the IKK (inhibitor of NFkB kinase) complex and the serine/ a clean genetic dissection of individual functions difficult. threonine kinase RIP-1. This protein complex shuttles from the cytoplasm to the nucleus, where NEMO is sumoylated.36 This sumoylation is facilitated by PIDD,8 although at the moment it is PIDD IN DNA REPAIR, CANCER AND DRUG RESISTANCE not clear whether PIDD directly influences this reaction, recruits Yet another complex containing PIDD was identified by large-scale ligases for sumoylation or prevents desumoylation that has IP and mass spectrometry analysis.9 Within this complex, PIDD recently been shown to rely on SENP2.37 Sumoylated NEMO is interacts in the nucleus with components of the replication factor subsequently bound by ATM (ataxia telangiectasia mutated) and complex 5 (RFC5) and proliferating cell nuclear antigen (PCNA) in phosphorylated at Serine-85.38 This phosphorylation is required response to UV-IR. This leads to detachment of the CDK-inhibitor for mono-ubiquitination by cIAP1 and export back to the p21, bound to PCNA, and subsequent degradation of p21 cytoplasm, as cells harboring a phosphorylation mutant of NEMO considered as a prerequisite for DNA repair in response to UV-IR. are deficient in both ubiquitination and nuclear export, but not Additionally, PIDD positively influences mono-ubiquitination of sumoylation.39 PCNA, thereby facilitating recruitment of the low fidelity repair Recently, several publications show further evidence of a highly DNA polymerase Z to chromatin. How exactly PIDD promotes complex mode of regulation leading to activation of IKK in ubiquitination of PCNA remains unknown, as well as whether the response to DNA damage. Immediately after the damage, Poly mechanism involves similar posttranslational modifications facili- (ADP-ribose) polymerase-1 (PARP1) becomes activated and poly- tated by PIDD on NEMO. Of note, PIDD-deficiency sensitized the ADP-ribosylated, which serves as platform for the recruitment of human keratinocyte cell line HaCaT to UV-C-mediated cell death ATM and protein inhibitor of activated STAT-4 (PIASy).40 In this as well as primary mouse keratinocytes to the lethal effects of the signalosome, PIASy acts as a sumo ligase for NEMO, leading to the more physiological UV-B irradiation in vivo. It will be interesting to retention of sumoylated NEMO in the nucleus.41 Phosphorylation see whether this effect culminates in reduced rates of mutational by ATM bound to NEMO in turn facilitates NEMO export back into load and/or altered latency of UV-driven skin cancer. the cytoplasm.38 Additionally, ATM is also exported to the The PIDD interaction partner caspase-2 is implicated to act as a cytoplasm in a Ca2 þ dependent manner, where it binds TRAF6.42 tumor suppressor in a model of B-cell lymphomas.31 However, This in turn leads to TRAF6 and Ubc13-dependent generation of until now there are no reports whether PIDD has a similar role in K63-linked ubiquitin chains on TRAF6 and ELKS. Ubiquitinated tumorigenesis as caspase-2 or whether the tumor suppressor TRAF and ELKS serve as a platform for the recruitment of TAK1 by function of caspase-2 depends on PIDD. Our own studies confirm TAB2/3, which then phosphorylates and activates IKKb.42 --44 At the tumor suppression by caspase-2 in response to aberrant c-Myc same time, sumoylated NEMO is mono-ubiquitinated and forms expression but this effect was independent of PIDD, at least in this part of a complex with IKKa, IKKb and ELKS that is essential for IKK model system (Manzl et al., submitted). The latter observation activation. This complex formation is dependent on binding of would be in line with a role in low fidelity DNA repair responses, NEMO to a yet to be identified protein. If any of these molecular such as non-homologous end-joining (NHEJ) also triggered in events also involves PIDD remains to be established. response to oncogenic stress.47 Of note here, PIDD was also found Studies mentioned above using RNA interference and over- to interact with the catalytic subunit of DNA-activated protein expression as well as our own studies using PIDD-deficient cells kinase (DNA-PK),9 but the physiological relevance of this observa- show significant defects in NFkB activation in response to tion remains to be reinvestigated. genotoxic stress, but not TNF, at least in tissue culture (Bock Expression analysis of various oral squamous cell carcinomas et al., submitted). This seemingly makes PIDD one of the few revealed an association between the apoptotic index and genes that are exclusively required for NFkB activation in response PIDD expression, in line with its proposed role in apoptosis.48 to DNA damage, but the analysis of cytokine release upon Surprisingly, no correlation between p53 status and PIDD systemic challenge of mice with IR suggests that PIDD may still expression was found, arguing for p53-independent PIDD expres- have a role in this process (Bock et al., submitted). Surprisingly, as sion changes in these tumors. Another, although indirect, cue on mentioned before, PIDD-deficient cells and mice show no the role of PIDD in cancer was shown in cell lines or primary cells difference in cell survival in response to DNA damage.16,28 This derived from patients with high-risk myelodysplastic syndrome or is in stark contrast to other mouse models lacking genes required acute myeloid leukemia.49 Those cells are characterized by for NFkB activation, which generally show decreased survival and constitutive NFkB activation that is essential for their survival, as frequently die in utero.45 Furthermore, different models of DNA blocking NFkB activation by ATM inhibition leads to apoptosis. In damage-induced cancer formation failed to show any signs of the model proposed by the authors, NEMO is activated by nuclear PIDD-dependency for onset and severity of disease (Bock et al., PIDD and ATM, thereby triggering NFkB signalling. submitted, Manzl et al., submitted). Interestingly, also absence of Furthermore, expression of PIDD was upregulated in the PARP1, limiting DNA damage-dependent activation of NFkB, fails testicular germ cell carcinoma cell line NT2/D1 after cisplatin to modulate IR-driven tumorigenesis, but, as loss of PIDD, affected treatment, suggesting a role for PIDD in the cell death response cytokine release in response to acute IR challenge in vivo (Bock triggered by this drug, as these tumors are frequently cured by et al., submitted). These results suggest that NFkB activation in cisplatin-based chemotherapy.50 Consistently, in a mouse model response to DNA damage is not sufficient to protect cells from of lung cancer, PIDD was shown to be highly upregulated in apoptosis or increase repair efficacy in premalignant cells and in tumors in response to in vivo cisplatin treatment.51 Surprisingly, the end has no significant influence on cancer initiation. We however, PIDD overexpression in turn facilitated resistance to propose it mainly orchestrates cytokine production, as during an cisplatin treatment. Hence, it will be interesting to see whether immune response, but do not rule out that it may still be high levels of PIDD associate with treatment failure in patients important for tumor cell survival, once disease is established. In suffering from lung cancer or testicular germ cell carcinoma. fact, it is well known that different inflammation-induced cancers In a subsequent study it was proposed that PIDD-driven drug are highly dependent on NFkB, mainly by constant secretion of resistance to cisplatin might be caused by sustained p53 prosurvival and proliferative cytokines as well as production of activation and increased p21 levels, noted upon PIDD over- prosurvival molecules.46 However, direct evidence that DNA expression, because of induction of a positive feedback loop.52 damage triggered tumor formation is dependent on NFkBis This loop seems to be initiated by PIDDosome-dependent Mdm2

& 2012 Macmillan Publishers Limited Oncogene (2012) 4733 --4739 PIDDosomes and the DNA damage response FJ Bock et al 4738 cleavage at D367, removing the C-terminal RING domain required 5 Lin Y, Ma W, Benchimol S. Pidd, a new death-domain-containing protein, is for its E3-ligase function and DC-Mdm2/p53 interaction-depen- induced by p53 and promotes apoptosis. Nat Genet 2000; 26: 122 --127. dent stabilization of p53. Although intriguing and in line with a 6 Telliez JB, Bean KM, Lin LL. LRDD, a novel leucine rich repeat and death domain discussed role of caspase-2 in cell cycle arrest,31 a number of containing protein. Biochim Biophys Acta 2000; 1478: 280 --288. inconsistencies remain. For example, why and how does small 7 Tinel A, Janssens S, Lippens S, Cuenin S, Logette E, Jaccard B et al. Autoproteolysis interfering RNA against PIDD or caspase-2, that shortens the time of PIDD marks the bifurcation between pro-death caspase-2 and pro-survival to p53 degradation because of lack of Mdm2 cleavage, sensitize NF-kappaB pathway. Embo J 2007; 26: 197 --208. cells to DNA damage-mediated apoptosis after doxorubicin 8 Janssens S, Tinel A, Lippens S, Tschopp J. PIDD mediates NF-kappaB activation in response to DNA damage. Cell 2005; 123: 1079 --1092. treatment? Presumably, the argument would be that less effective 9 Logette E, Schuepbach-Mallepell S, Eckert MJ, Leo XH, Jaccard B, Manzl C et al. induction of p21 should sensitize to cell death induction, yet PIDD orchestrates translesion DNA synthesis in response to UV irradiation. Cell impaired p21 induction was not formally demonstrated in Death Differ 2011; 18: 1036 --1045. PIDDosome defective cells. A recent study also suggests that 10 Tinel A, Tschopp J. The PIDDosome, a protein complex implicated in activation of knockdown of caspase-2 can negatively affect p21 levels after caspase-2 in response to genotoxic stress. Science 2004; 304: 843 --846. DNA damage, but this effect did not require the proteolytic 11 Pick R, Badura S, Bosser S, Zornig M. Upon intracellular processing, the C-terminal activity of caspase-2, nor PIDD or RAIDD, suggesting translational death domain-containing fragment of the p53-inducible PIDD/LRDD protein control.53 Nonetheless, sensitization of PIDD-knockdown cells to translocates to the nucleoli and interacts with nucleolin. Biochem Biophys Res cell death was also reported after exposure to UV, but there it Commun 2006; 349: 1329 --1338. 9 12 Cuenin S, Tinel A, Janssens S, Tschopp J. p53-induced protein with a death correlated with impaired p21 degradation. Furthermore, doxo- domain (PIDD) isoforms differentially activate nuclear factor-kappaB and caspase- rubicin-driven cell death in cells wild type for p53 usually depends 2 in response to genotoxic stress. Oncogene 2008; 27: 387 --396. on p53-driven expression of PUMA and/or Noxa, pro-apoptotic 13 Huang L, Han D, Yang X, Qin B, Ji G, Yu L. PIDD4, a novel PIDD isoform without the proteins of the Bcl-2 family that should then be activated less LRR domain, can independently induce cell apoptosis in cytoplasm. Biochem effectively as well, culminating in reduced death rates in the Biophys Res Commun 2011; 407:86--91. absence of the PIDDosome, but the opposite was observed in 14 Heinz LX, Rebsamen M, Rossi DC, Staehli F, Schroder K, Quadroni M et al. The U2OS cells lacking PIDD.52 Analysis of primary lymphocytes and death domain-containing protein Unc5CL is a novel MyD88-independent MEF lacking PIDD or caspase-2, however, did not provide evidence activator of the pro-inflammatory IRAK signaling cascade. Cell Death Differ 2011 for differences in cell survival in response to several DNA- (e-pub ahead of print 9 December 2011; doi:10.1038/cdd.2011.147). 16,28 15 Rosenblum JS, Blobel G. Autoproteolysis in nucleoporin biogenesis. Proc Natl Acad damaging agents. Sci U S A 1999; 96: 11370 --11375. 16 Manzl C, Krumschnabel G, Bock F, Sohm B, Labi V, Baumgartner F et al. Caspase-2 activation in the absence of PIDDosome formation. J Cell Biol 2009; FINAL REMARKS 185: 291 --303. Despite a growing number of research articles, the exact 17 Tinel A, Eckert MJ, Logette E, Lippens S, Janssens S, Jaccard B et al. Regulation of physiological role of PIDD is still poorly defined. Although PIDD PIDD auto-proteolysis and activity by the molecular chaperone Hsp90. Cell Death was shown to be a part of several different protein complexes Differ 2011; 18: 506 --515. mediating various signalling pathways when overexpressed, the 18 Li J, Yuan J. Caspases in apoptosis and beyond. Oncogene 2008; 27: 6194 --6206. absence of a phenotype of PIDD-deficient cells and mice so far 19 Pop C, Salvesen GS. Human caspases: activation, specificity, and regulation. J Biol Chem 2009; 284: 21777 --21781. excludes an essential role of physiological importance within 20 Read SH, Baliga BC, Ekert PG, Vaux DL, Kumar S. A novel Apaf-1-independent those pathways. It is therefore likely that PIDD is either highly putative caspase-2 activation complex. J Cell Biol 2002; 159: 739 --745. redundant with other proteins, is only required for fine-tuning of 21 Krumschnabel G, Sohm B, Bock F, Manzl C, Villunger A. The enigma of caspase-2: certain pathways, which could not be clearly defined in past work, the laymen’s view. Cell Death Differ 2009; 16: 195 --207. or, alternatively, may rather be involved in cellular responses 22 Duan H, Dixit VM. RAIDD is a new ‘death’ adaptor molecule. Nature 1997; 385:86--89. secondary to or different from the DNA-damage response. 23 Niizuma K, Endo H, Nito C, Myer DJ, Kim GS, Chan PH. The PIDDosome mediates Production of inflammatory cytokines downstream of NFkB delayed death of hippocampal CA1 neurons after transient global cerebral activation or participation in the sensing of other types of DNA ischemia in rats. Proc Natl Acad Sci U S A 2008; 105: 16368 --16373. or damage, for example, in the innate immune response, may be 24 Hasegawa H, Yamada Y, Tsukasaki K, Mori N, Tsuruda K, Sasaki D et al. LBH589, a deacetylase inhibitor, induces apoptosis in adult T-cell leukemia/lymphoma considered as well. Clearly more and refined research is required cells via activation of a novel RAIDD-caspase-2 pathway. Leukemia 2011; 25: to define the biological significance of PIDD. 575 --587. 25 Vakifahmetoglu H, Olsson M, Orrenius S, Zhivotovsky B. Functional connection between p53 and caspase-2 is essential for apoptosis induced by DNA damage. CONFLICT OF INTEREST Oncogene 2006; 25: 5683 --5692. The authors declare no conflict of interest. 26 Olsson M, Vakifahmetoglu H, Abruzzo PM, Hogstrand K, Grandien A, Zhivotovsky B. DISC-mediated activation of caspase-2 in DNA damage-induced apoptosis. Oncogene 2009; 28: 1949 --1959. ACKNOWLEDGEMENTS 27 Sidi S, Sanda T, Kennedy RD, Hagen AT, Jette CA, Hoffmans R et al. Chk1 suppresses a caspase-2 apoptotic response to DNA damage that bypasses p53, This review is dedicated to the late Ju¨rg Tschopp, who spearheaded research on Bcl-2, and caspase-3. Cell 2008; 133: 864 --877. PIDD, a great scientist, colleague and mentor. Research on PIDD in our laboratory is 28 Kim IR, Murakami K, Chen NJ, Saibil SD, Matysiak-Zablocki E, Elford AR et al. DNA supported by grants from the Austrian Science Fund (FWF; Y212-B12 and SFB021), damage- and stress-induced apoptosis occurs independently of PIDD. Apoptosis EU-FP6 RTN ‘Apoptrain’, the Tyrolean Science Fund (TWF) and the ‘Krebshilfe-Tirol’. 2009; 14: 1039 --1049. 29 Bergeron L, Perez GI, Macdonald G, Shi L, Sun Y, Jurisicova A et al. Defects in regulation of apoptosis in caspase-2-deficient mice. Genes Dev 1998; 12: REFERENCES 1304 --1314. 1 Jackson SP, Bartek J. The DNA-damage response in human biology and disease. 30 Ho LH, Read SH, Dorstyn L, Lambrusco L, Kumar S. Caspase-2 is required for cell Nature 2009; 461: 1071 --1078. death induced by cytoskeletal disruption. Oncogene 2008; 27: 3393 --3404. 2 Vousden KH, Lane DP. p53 in health and disease. Nat Rev Mol Cell Biol 2007; 31 Ho LH, Taylor R, Dorstyn L, Cakouros D, Bouillet P, Kumar S. A tumor suppressor 8: 275 --283. function for caspase-2. Proc Natl Acad Sci U S A 2009; 106: 5336 --5341. 3 Meek DW. Tumour suppression by p53: a role for the DNA damage response? Nat 32 Berube C, Boucher LM, Ma W, Wakeham A, Salmena L, Hakem R et al. Apoptosis Rev Cancer 2009; 9: 714 --723. caused by p53-induced protein with death domain (PIDD) depends on the death 4 Olsson A, Manzl C, Strasser A, Villunger A. How important are post-translational adapter protein RAIDD. Proc Natl Acad Sci U S A 2005; 102: 14314 --14320. modifications in p53 for selectivity in target-gene transcription and tumour 33 Janssens S, Tschopp J. Signals from within: the DNA-damage-induced NF-kappaB suppression? Cell Death Differ 2007; 14: 1561 --1575. response. Cell Death Differ 2006; 13: 773 --784.

Oncogene (2012) 4733 --4739 & 2012 Macmillan Publishers Limited PIDDosomes and the DNA damage response FJ Bock et al 4739 34 Miyamoto S. Nuclear initiated NF-kappaB signaling: NEMO and ATM take center mitogen-activated protein kinase (MAPK)/MAPK-activated protein 2 responses to stage. Cell Res 2011; 21: 116 --130. DNA damage. Mol Cell Biol 2011; 31: 2774 --2786. 35 Hayden MS, West AP, Ghosh S. NF-kappaB and the immune response. Oncogene 44 Wu ZH, Wong ET, Shi Y, Niu J, Chen Z, Miyamoto S et al. ATM- and NEMO- 2006; 25: 6758 --6780. dependent ELKS ubiquitination coordinates TAK1-mediated IKK activation in 36 Huang TT, Wuerzberger-Davis SM, Wu ZH, Miyamoto S. Sequential modiﬁcation of response to genotoxic stress. Mol Cell 2010; 40: 75 --86. NEMO/IKKgamma by SUMO-1 and ubiquitin mediates NF-kappaB activation by 45 Karin M, Cao Y, Greten FR, Li ZW. NF-kappaB in cancer: from innocent bystander genotoxic stress. Cell 2003; 115: 565 --576. to major culprit. Nat Rev Cancer 2002; 2: 301 --310. 37 Lee MH, Mabb AM, Gill GB, Yeh ET, Miyamoto S. NF-kappaB induction of the 46 Ben-Neriah Y, Karin M. Inﬂammation meets cancer, with NF-kappaB as the SUMO Protease SENP2: a negative feedback loop to attenuate cell survival matchmaker. Nat Immunol 2011; 12: 715 --723. response to genotoxic stress. Mol Cell 2011; 43: 180 --191. 47 Heinen CD, Schmutte C, Fishel R. DNA repair and tumorigenesis: lessons from 38 Wu ZH, Shi Y, Tibbetts RS, Miyamoto S. Molecular linkage between the kinase ATM hereditary cancer syndromes. Cancer Biol Ther 2002; 1: 477 --485. and NF-kappaB signaling in response to genotoxic stimuli. Science 2006; 311: 48 Bradley G, Tremblay S, Irish J, MacMillan C, Baker G, Gullane P et al. The expression 1141 --1146. of p53-induced protein with death domain (Pidd) and apoptosis in oral squamous 39 Jin HS, Lee DH, Kim DH, Chung JH, Lee SJ, Lee TH. cIAP1, cIAP2, and cell carcinoma. Br J Cancer 2007; 96: 1425 --1432. XIAP act cooperatively via non-redundant pathways to regulate genotoxic 49 Grosjean-Raillard J, Tailler M, Ades L, Perfettini JL, Fabre C, Braun T et al. stress-induced nuclear factor-kappaB activation. Cancer Res 2009; ATM mediates constitutive NF-kappaB activation in high-risk myelodysplastic 69: 1782 --1791. syndrome and acute myeloid leukemia. Oncogene 2009; 28: 1099 --1109. 40 Stilmann M, Hinz M, Arslan SC, Zimmer A, Schreiber V, Scheidereit C. A nuclear 50 Kerley-Hamilton JS, Pike AM, Li N, DiRenzo J, Spinella MJ. A p53-dominant poly(ADP-ribose)-dependent signalosome confers DNA damage-induced IkappaB transcriptional response to cisplatin in testicular germ cell tumor-derived human kinase activation. Mol Cell 2009; 36: 365 --378. embryonal carcinoma. Oncogene 2005; 24: 6090 --6100. 41 Mabb AM, Wuerzberger-Davis SM, Miyamoto S. PIASy mediates NEMO sumoyla- 51 Oliver TG, Mercer KL, Sayles LC, Burke JR, Mendus D, Lovejoy KS et al. Chronic tion and NF-kappaB activation in response to genotoxic stress. Nat Cell Biol 2006; cisplatin treatment promotes enhanced damage repair and tumor progression in 8: 986 --993. a mouse model of lung cancer. Genes Dev 2010; 24: 837 --852. 42 Hinz M, Stilmann M, Arslan SC, Khanna KK, Dittmar G, Scheidereit C. A cytoplasmic 52 Oliver TG, Meylan E, Chang GP, Xue W, Burke JR, Humpton TJ et al. Caspase-2- ATM-TRAF6-cIAP1 module links nuclear DNA damage signaling to ubiquitin- mediated cleavage of Mdm2 creates a p53-induced positive feedback loop. mediated NF-kappaB activation. Mol Cell 2010; 40:63--74. Mol Cell 2011; 43:57--71. 43 Yang Y, Xia F, Hermance N, Mabb A, Simonson S, Morrissey S et al. A cytosolic 53 Sohn D, Budach W, Janicke RU. Caspase-2 is required for DNA damage-induced ATM/NEMO/RIP1 complex recruits TAK1 to mediate the NF-kappaB and p38 expression of the CDK inhibitor p21(WAF1/CIP1). Cell Death Differ 2011; 18:1664-- 1674.

& 2012 Macmillan Publishers Limited Oncogene (2012) 4733 --4739